1. Field of the Invention
The present invention relates to an integral multilayer analytical element for use in the analysis of an ammonia-forming substrate in liquid samples, and more particularly to an integral multilayer analytical element suitable for use in the analysis (assay) of an ammonia-forming substrate, such as, creatinine, urea, etc., in body fluids, such as, blood, urine, lymph fluid, etc. without being affected by intrinsic ammonia.
2. Description of Prior Arts
The assaying of ammonia-forming substrates, such as, creatinine, urea, etc., is very important in the diagnosis of diseases, such as renopathy, the inspection of the progress of diseases and the inspection of the function of kidney.
A typical method for analyzing ammonia-forming substrates is a method comprising forming ammonia from the substrates and determining the amount of formed ammonia. This analytical method utilizing the conversion of the substrates into ammonia has been widely used as a method which is generally called a wet process or a solution method.
In the above operation for forming ammonia from the ammonia-forming substrates, method usually used is forming ammonia by the action of an enzyme. For instance, in the analysis of creatinine in body fluids, a method is utilized in which creatinine in the body fluids is specifically hydrolyzed into ammonia and N-methyl hydantoin by using creatinine deiminase (EC 3.5.4.21). In the analysis of blood urea nitrogen (hereinafter referred to as BUN), a method is used in which urea is hydrolyzed into ammonia and carbon dioxide by using urease as a catalyst.
For determining the amount of ammonia formed, various methods known as ammonia-assaying methods can be used. For instance, a method in which the formed an ammonia is determined by means of ammonia ion-selective electrode, and a method in which the color of a pH indicator is changed by the ammonia formed and the degree of color change is measured to thereby determine the amount of ammonia are used. Also used is a method in which the formed ammonia is coupled with .alpha.-ketoglutaric acid in the presence of glutamate dehydrogenase (EC 1.4.1.3) and NADH (nicotinamide adenine dinucleotide of the reduced type) to form L-glutamic acid and water. The amount of NAD.sup.+ (nicotinamide adenine dinucleotide of the oxidized type) to be formed or the amount of NADH to be reduced by the conversion of NADH into NAD.sup.+ is determined by means of fluorometry or photometry utilizing near-ultraviolet light at a wavelength of 340 nm. Another method is used in which NADPH (nicotinamide adenine dinucleotide phosphate of the reduced type) is used in place of said NADH, and the amount of NADP.sup.+ (nicotinamide adenine dinucleotide phosphate of oxidized type) to be formed or the amount of NADPH to be reduced by the conversion of NADPH into NADP.sup.+ is determined.
The analytical methods for the ammonia-forming substrates comprising the aforementioned two operations are described in literature, such as, Analytical Chemistry, 46, 246(1974); Clinica Chemica Acta, 18, 409(1967); Clinical Chemical Analysis III Nitrogen-containing Component (written in Japanese), the second edition, pages 13-14 and 67-87 (1979) (Tokyo Kagaku Dojin Ltd., Japan); and Clinical Inspection, Vol. 5, No. 6, pages 387-391 (1961) written in Japanese.
Ammonia (intrinsic ammonia) is frequently present as ammonium ion in the free form in body fluids to be analyzed, and the error caused by the intrinsic ammonia is non-negligible. Accordingly, it is necessary to eliminate the influence of the intrinsic ammonia on the accurate determination of the ammonia forming substrates, and methods have been proposed for removing intrinsic ammonia.
An example of an analytical method for ammonia forming substrates including an operation for previously removing intrinsic ammonia is an automatic continuous flowing method for creatinine analyte disclosed in Japanese Patent Provisional Publication No. 58(1983)-5198 in which an operation comprising reacting a sample with a reagent (e.g., a reagent consisting of glutamate dehydrogenase, .alpha.-ketoglutamic acid and NADH) capable of removing intrinsic ammonia and dialyzing any unreacted creatinine contained in the reaction mixture by a dialyzer is carried out as a pre-step in an operation for forming ammonia from creatinine.
Methods for pretreating an analyte are disclosed in Japanese Patent Provisional Publication Nos. 59(1984)-21398 and 59(1984)-31689 comprising using NADP reductase (or NAD reductase), restoring NADP.sup.+ (or NAD.sup.+) formed as a by-product to the original NADPH (or NADH) and recycling for use in a method for eliminating intrinsic ammonia by reacting NADPH (or NADH), glutamate dehydrogenase and .alpha.-ketoglutamic acid with the intrinsic ammonia. In the analysis of the ammonia-forming substrates by using said pretreating method, an enzyme capable of forming ammonia from the substrate is added to the pretreated analyte and the formed ammonia is determined by using a reagent system comprising NADH (or NADPH), glutamate dehydrogenase and .alpha.-ketoglutamic acid as described in Japanese Patent Provisional Publication Nos. 59(1984)-31696, 59(1984)-31698 and 59(1984)-31700.
The above-described analytical methods have been carried out by methods conventionally called wet process or a solution method, or they have been proposed with a view to conducting a wet process. Nevertheless, an analytical method which can be performed with only a small amount of a sample and can be easily operated with high accuracy, has been highly desired by medical persons, such as, medical doctors in clinical tests. Therefore, dry analytical methods which are easy to handle, are now used in place of the conventional wet analytical methods. As an analytical element for the ammonia-forming substrate in the dry analytical method, a test piece for the analysis of blood urea nitrogen (BUN) is disclosed in U.S. Pat. No. 3,011,874. Further, analytical methods have been developed analytical methods using an integral multilayer analytical element (hereinafter referred to simply as analytical element) which can be easily operated with high accuracy, and the improvements in the element are being made.
Examples of integral multilayer analytical elements which can be used for the analysis of the ammonia-forming substrates include the element disclosed in Japanese Patent Publication No. 58(1983)-19062 and the elements for the analysis of ammonia or the ammonia-forming substrate described in Japanese Patent Provisional Publication Nos. 58(1983)-77660 and 58(1983)-77661. These analytical elements comprise basically laminating an ammonia indicator layer capable of undergoing a detectable change by contact with ammonia, a barrier layer which functions as a liquid barrier and allows gaseous ammonia to be passed therethrough, a reaction layer containing a reagent capable of forming ammonia by the reaction with an ammonia-forming substrate and a porous spreading layer in this order onto a liquid-impermeable, light-transmissive support. However, these analytical elements can not eliminate the influence of ammonia (intrinsic ammonia) present in body fluids. Accordingly, it is difficult to determine accurately the ammonia-forming substrate by these analytical elements.
The method for removing intrinsic ammonia described in the aforementioned Japanese Patent Provisional Publication No. 58(1983)-5198, which must use a dialysis device for discharging a product of the reaction between a reagent and intrinsic ammonia to the outside of the reaction system, is generally applied to a wet analytical method and cannot be used, as, such for the dry analytical method using said integral multilayer analytical element. In the method described in the aforementioned Japanese Patent Provisional Publication No. 59(1984)-21398, complicated operations comprising reacting intrinsic ammonia, measuring the OD of a blank and adding creatinine deiminase are carried out and then creatinine is determined by a reaction for forming ammonia from creatinine.